Geographical distribution and phoretic associations of the viviparous nematode Tokorhabditis atripennis with Onthophagus dung beetles in Japan

Abstract Viviparity is generally considered to be rare in animals. In nematodes, only six species of Rhabditida are viviparous. Five of these species have been identified in association with Onthophagus dung beetles, with Tokorhabditis atripennis being repeatedly isolated from the dung beetle Onthophagus atripennis in Japan. T. atripennis is easy to culture in a laboratory setting, and its host, O. atripennis, is distributed all over Japan. Therefore, T. atripennis is an ideal candidate for ecological and evolutionary studies on viviparity. However, the extent of their distribution and relationship with dung beetles, as well as habitats, remain unclear. In the present study, we conducted field surveys and successfully isolated 27 strains of viviparous nematodes associated with tunneler dung beetles from various regions of Japan, all of which were identified as T. atripennis. T. atripennis exhibited a strong association with Onthophagus dung beetles, especially O. apicetinctus and O. atripennis. And it was predominantly found in specific anatomical locations on the beetle bodies, such as the ‘groove between pronotum and elytron’ and the ‘back of the wings’. Our findings suggest that Onthophagus species are the primary hosts for T. atripennis, and T. atripennis exhibits a close relationship with the living environments of tunneler beetles. This association may play a significant role in the evolution of viviparity in nematodes.

Throughout their evolutionary history, animals have developed diverse reproductive strategies, adapting to a wide range of ecological systems.Among animals, viviparity stands out as one of the most prevalent reproductive modes, primarily employed as a strategy to increase the body size of offspring before delivery rather than their quantity.Viviparity is an incubation mode in which embryonic development occurs within the reproductive system (ovary or sexual duct), body cavity (coelom, pseudocoel, or hemocoel), parental tissues, or tissue-like layers (parenchyma, mesohyl, or mesoglea) with nutrient supply, resulting in live births (Ostrovsky et al., 2016).It has evolved independently at least 160 times (Blackburn, 1999), suggesting that certain environmental factors influence the evolution of viviparous traits in these animals.However, empirical research demonstrating its specific role and the driving forces behind its evolution remains largely lacking in many animal species.
Although nematodes are generally oviparous, females of some nematode species retain fertilized eggs and are described as viviparous.Viviparity in the Association between viviparous nematode and dung beetles: Ikeda et al.
Rhabditida is typically facultative, either manifesting as endotokia matricida ("bagging"; also "aparity," sensu Sudhaus 1976) or as live birth of young that hatch from rigid eggs in utero (Johnigk and Ehlers, 1999;Chen and Caswell-Chen, 2004;Vigne et al., 2021).However, only six species within the Rhabditida appear to meet the definition of obligate viviparity, which obligately retains fertilized eggs with embryonic growth and nutritional provision (Herrmann et al., 2013;Kanzaki et al., 2017Kanzaki et al., , 2021;;Ragsdale et al., 2022;Yamashita et al., 2023).Of these, five have been found to be associated with Onthophagus dung beetles, with the exception of Tokorhabditis tufae, which was discovered in the alkaline, hypersaline, and arsenic-rich environment of Mono Lake, California (Shih et al., 2019).One such species, Tokorhabditis atripennis was discovered in association with the dung beetle Onthophagus atripennis (Ragsdale et al., 2022).Onthophagus dung beetles engage in tunneling beneath animal dung, crafting brood balls from the dung that serve as nourishment and nesting sites for their larvae, a behavior indicative of tunnelers (Bornemissza, 1969;Bornemissza, 1976;Cambefort and Hanski, 1991).This suggests that the viviparous nematode T. atripennis is a dung beetle-phoretic species dwelling within fecal environments, including brood balls, and that its reproductive mode is an adaptation to these specialized habitats.
Given that T. atripennis is readily found in association with Onthophagus dung beetles in the field and can be cultured in the laboratory, the nematode-dung beetle system is an excellent model for studying the evolution of animal reproduction.However, because of the lack of a comprehensive sampling survey of T. atripennis, the following essential questions remain unanswered: (1) the preferred dung beetle taxon for T. atripennis as a host, (2) whether T. atripennis exhibits phoretic behavior with dung beetles (with the potential for parasitism or accidental association), and (3) the geographical range of its distribution.Although viviparity in T. atripennis appears to be an adaptation to dung beetle habitats, the lack of ecological information on this nematode makes it challenging to uncover the potential factors behind its viviparous reproductive strategy.
In this study, we aimed to investigate the relationship between T. atripennis and dung beetles and to gather ecological information regarding their distribution.We conducted fieldwork and collected data on 615 dung beetles from 12 Japanese prefectures.Subsequently, we performed dissections and investigated the following aspects: 1) The rate of association between T. atripennis and each dung beetle species; 2) Specific anatomical locations within dung beetle bodies where nematodes were found; 3) Regional variations in the prevalence of T. atripennis associations with dung beetles across Japan.
The collected beetles were individually stored in plastic cases with humid tissue paper until dissection.Before dissection, each beetle's viability was confirmed, and they were observed under a stereo microscope for species and sex identification following the illustration reference, "Scarab beetles of Japan" (Kawai et al., 2008).Most beetles were able to survive for up to one week using this sampling method.However, if any beetles died, they were excluded from further study.
The dung beetles were dissected to verify the presence or absence of phoretically associated nematodes.We identified six distinct body parts of the nematode: 1) the entire body surface, 2) the dorsum of the wings, 3) the pronotum-elytron groove, 4) the male genitalia and testis, 5) ovary, and 6) the pronotum-front groove and the anterior regions for locating the phoretic nematodes (Fig. 1).We carefully rinsed the surface of the dung beetles with ion exchange water (IEW) on a Syracuse watch glass and looked for nematodes under a dissecting microscope.Subsequently, we dissected the beetle elytron and examined the dorsal area as well as the groove between the pronotum and the elytron in the presence of nematodes.Next, we removed and examined the male genitalia, testes, and ovaries for the presence of nematodes.Then, we detached the heads of dung beetles from the pronotum and observed the presence of nematodes.
Nematode isolation: Once nematodes were found during the dissection of dung beetles, we transferred them onto nematode growth medium (NGM) (Brenner, 1974), as well as onto the NGM + dog food medium (DFM: 20 g crushed dog food, 4 g agar and IEW were poured until the total volume became 200 ml, autoclaved and solidified) seeded with E. coli OP50 and incubated them at 25 °C (Hara et al., 1981;Ogura and Mamiya, 1989).Two to three days after being transferred to NGM or DFM plates, most freeliving nematodes reached the adult stage, allowing us to distinguish their reproductive modes as either oviparous or viviparous.Viviparous nematodes were sterilized using a 10% SDS solution and established as laboratory strains, except for the strains of SHR   that were just washed in IEW.Although oviparous nematodes were collected in this study, they were not identified as strains.
The obtained DNA fragments were purified from agarose gels with NucleoSpin ® Gel and PCR Clean-up (Macherey-Nagel, Germany) or FastGene Gel/ PCRExtraction kit (NIPPON Genetics Co., Ltd, Japan).Samples were submitted to Hokkaido System Science Co. (Sapporo, Japan) or Macrogen Japan Corp. for sequencing from both strands using the same PCR primers.The sequences obtained were confirmed and edited manually using a Serial Cloner (ver.2-6-1) and identified to the species level based on the results of the Basic Local Alignment Search Tool (BLAST) analysis.Isolated nematodes were considered potentially distinct species if they exhibited <99% sequence similarity to their nearest neighbors.Sequence data were deposited in the NCBI for Biotechnology Information GenBank database.
Association between viviparous nematode and dung beetles: Ikeda et al.
Statistical analysis: Significant differences in the percentages of dung beetles associated with viviparous nematodes among regions, beetle genera, and species were determined.First, a Fisher's exact test was performed using the R package in Jupyter Lab.When significant differences were detected by the Fisher's exact test, Tukey's WSD was performed for multiple comparisons at P < 0.05.

Results
All isolated free-living viviparous nematodes were T. atripennis: A total of 615 dung beetles were collected from 12 prefectures and classified into 30 species across six genera (Table S2).Most of the nematodes associated with dung beetles were freeliving nematodes with oviparous reproduction (data not shown).This was evident when eggs were laid on plates seeded with E. coli OP50 after reaching adulthood.However, a subset of the isolated nematodes did not lay eggs, but released larvae, leading us to classify them as exhibiting viviparous reproduction.
All isolated viviparous nematodes were successfully cultured on plates, genomic DNA was isolated, and sequences of the LSU and SSU genes were obtained.In total, 27 viviparous nematode strains were obtained from individual dung beetles of eight species found in six prefectures (Fig. 2; Table 2).Upon comparing their LSU and SSU gene sequences, it was evident that all genes in the 27 strains were identical to those of T. atripennis.The NCBI accession numbers assigned to the gene sequences of each strain are shown in Table S3.

Discussion
The six genera of dung beetles collected in this study can be categorized into two ecological types: tunneler beetles (Copris, Caccobius, Onthophagus and Phelotrupes), which dig tunnels beneath animal dung to create nests, and dweller beetles (Aphodius and Ataenius), which establish nests directly on the dung (Bornemissza, 1969;Bornemissza, 1976;Cambefort and Hanski, 1991).We only observed an association between the viviparous nematode T. atripennis and tunneler beetles, especially Onthophagus beetles (Table 3).Among Onthophagus beetles, the association rate was the highest for O. apicetinctus, which was primarily distributed in the Yaeyama Islands (Fig. 2; Tables 3 and 5).Conversely, O. atripennis, a species distributed throughout Japan, exhibited a higher association rate than the other species in different regions.This suggests that T. atripennis may reside in specific structures of the living environment of tunneler beetles, such as brood balls, and that specific biotic or abiotic factors in such environments may have driven the evolution of viviparity in nematodes.Among the tunneler beetles, the association rates of T. atripennis with O. apicetinctus and O. atripennis were significantly higher than those with other dung beetles (Table 3).In addition, the phoretic association sites of T. atripennis appeared to vary between Onthophagus and other dung beetles (Table 4).This suggests that T. atripennis exhibits a preference for associating with Onthophagus beetles, showing a strong affinity for the two species, O. apicetinctus and O. atripennis, which could be considered the most suitable hosts for T. atripennis.The tendency of T. atripennis to attach to specific body parts of beetles suggests a symbiosis between T. atripennis and Onthophagus beetles.For example, dung beetles may act as carriers of nutrient-rich feces for T. atripennis, while T. atripennis could help create a favorable living environment for dung beetles.
Most nematodes described are oviparous; viviparous nematodes are considered rare.However, our study and previous studies have identified viviparous nematodes associated with Onthophagus dung beetles in different regions worldwide (Herrmann et al., 2013;Kanzaki et al., JOURNAL OF NEMATOLOGY 2017, 2021;Ragsdale et al., 2022).Therefore, viviparous nematodes appear to have a broader distribution than previously thought.This suggests that the evolution of viviparity in nematodes may be related to the living environment of specific Onthophagus species.Further research is needed to clarify the ecological function and driving force of viviparity in relation to Onthophagus species.Since some researchers have reported nematode behavior in dung environments recreated in the laboratory (Kühne, 1996;Ledón-Rettig et al., 2018), these studies and our findings are expected to contribute to a better understanding of the life cycles of viviparous nematodes in natural environments and their interactions with other organisms.

Figure 2 :
Figure 2: Geographical distribution of Tokorhabditis atripennis in Japan.The numbers in the figure are the proportions of test beetles that were associated with T. atripennis.

Figure 1 :
Figure 1: The body parts of beetles examined the number of Tokorhabditis atripennis associated.

Table 1 :
The number of beetles collected for each attractant and collection site, with the exception of pit-hole traps.

Table 2 :
Information on all Tokorhabditis atripennis strains obtained in this study.
Different letters indicate significance differences (P < 0.05, Tukey WSD).Species names are written only when the beetles were associated.

Table S1 :
Primer list used in this study.